Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Abstract

XRCC4-null mice have a more severe phenotype than KU80-null mice. Here, we address whether this difference in phenotype is connected to nonhomologous end-joining (NHEJ). We used intrachromosomal substrates to monitor NHEJ of two distal doublestrand breaks (DSBs) targeted by I-SceI, in living cells. In xrcc4-defective XR-1 cells, a residual but significant end-joining process exists, which primarily uses microhomologies distal from the DSB. However, NHEJ efficiency was strongly reduced in xrcc4-defective XR-1 cells versus complemented cells, contrasting with KU-deficient xrs6 cells, which showed levels of end-joining similar to those of complemented cells. Nevertheless, sequence analysis of the repair junctions indicated that the accuracy of end-joining was strongly affected in both xrcc4-deficient and KU-deficient cells. More specifically, these data showed that the KU80/XRCC4 pathway is conservative and not intrinsically error-prone but can accommodate non-fully complementary ends at the cost of limited mutagenesis.double-strand break repair ͉ genome rearrangements D NA double-strand breaks (DSBs) are harmful lesions generated by a variety of endogenous or exogenous stresses, potentially leading to genomic rearrangement. Nonhomologous endjoining (NHEJ) is a prominent pathway for DSB repair (1). Canonical NHEJ involves the successive intervention of the KU80-KU70 heterodimer, DNA-PKcs-Artemis, and, finally, ligase IV (Lig4) associated with its cofactors XRCC4 and Cernunnos/Xlf (2, 3). KU-independent NHEJ (KU-alt) has been described in vitro in both acellular extracts and cultured cells (1, 4-6).Alternative, XRCC4-independent DSB repair pathways (XRCC4-alt) have also been described, using episomic plasmid in cultured cells, using pulse field gel electrophoresis, or in in vitro biochemical experiments (5-10). One hypothesis could propose that XRCC4 and KU are implicated in the same canonical NHEJ pathway, whereas the alternate pathway is independent of both KU and XRCC4. However, in transgenic mice, the inactivation of XRCC4 or Lig4 results in a more severe phenotype than the inactivation of KU (11), suggesting that XRCC4 might have an additional essential function; however, studies show that XRCC4 and Lig4 do not have roles outside of NHEJ, whereas in contrast, KU acts in other processes such as transcription, apoptosis, and responses to the cell microenvironment (12)(13)(14).Alternatively, these varying phenotypes in mice may actually result from differences in DSB repair efficiencies, indicating that defects in XRCC4 might be more deleterious for DSB repair than defects in KU. What challenges this hypothesis, however, is that substantial class switch recombination (CSR) has recently been shown to occur in mouse B cells without XRCC4, whereas no CSR was recorded in cells devoid of KU (15-18).The relative contributions of XRCC4 and KU80 versus the XRCC4-alt and KU-alt pathways, respectively, to DSB repair remain unclear in wild-type cells. Contrasting results were obtained in living cells, using an episomic plasm...

Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70

“…End-joining in the absence of C-NHEJ has been observed in the context of transiently transfected, linearized plasmid substrates and for AID or I-SceI-generated chromosomal DSBs in cells deficient for Ku80, XRCC4, or Lig4 (20)(21)(22)(23)(24)(25). In addition, XRCC4 or Lig4-deficient B cells undergo CSR at levels up to 50% those of WT B cells and, in contrast to CSR junctions in normal B cells, nearly all CSR junctions are MH-mediated (14,19).…”

Alternative endings

“…In the aforementioned article in PNAS by Guirouilh-Barbat et al (1) and in several recent papers (2-4), the characterization of an alternative, Ku-independent MMEJ mechanism in mammalian cells has been shown to be independent of LIG4 and XRCC4. In the article by Guirouilh-Barbat et al, joinings of I-SceIinduced DSBs were examined in XRCC4-deficient cells.…”

“…End-joining also is responsible for generating chromosomal translocations that are associated with various cancers. Work by GuirouilhBarbat et al (1) published in a recent issue of PNAS and by several other laboratories (2-4) has revealed that-in addition to the ''classical'' NHEJ pathway-there is a robust alternative pathway that also contributes to the generation of translocations.…”

Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency

“…ALT-EJ is independent of C-NHEJ factors and mediated by LIG1 and/or LIG3 among other factors (5-7). Repair junctions mediated by these EJ pathways show distinct patterns (6). For example, the repair junctions in C-NHEJ-deficient cells, which by definition, are mediated by ALT-EJ, show a greater frequency of deletion mutations (6).…”

“…Repair junctions mediated by these EJ pathways show distinct patterns (6). For example, the repair junctions in C-NHEJ-deficient cells, which by definition, are mediated by ALT-EJ, show a greater frequency of deletion mutations (6). Furthermore, the deletion junctions in C-NHEJdeficient cells also show frequent microhomology, which refers to short stretches of homology that bridge the DSB ends during EJ (6).…”